This invention refers to an aqueous base inoculant composition for seeds, particularly leguminosae, containing at least one species of nitrogen fixing microorganisms in plants, more particularly bacteria of Rhizobium or Bradyrhizobium genus, which provides the plants with increased nodulation capacity, and remains stable at room temperature, without decreasing the number of viable bacteria for extended periods of time.
The symbiotic relation between leguminous plants and bacteria of Rhizobium genus is very well known. Leguminous plants form a large group of plants to which vegetables of economic importance as soybean, lucerne, peanut, peas, etc., belong. The bacteria of Rhizobium genus are able to infect the roots of the plants, as they permeate into the radicular hairs and colonize the root, producing those characteristic xe2x80x9cnodulesxe2x80x9d. As a result of this symbiotic relation, plants can turn gaseous nitrogen into organic compounds of nitrogen, by means of the process described as nitrogen fixation.
When seeds of leguminosae are sowed in an area which has not been previously cultivated, or that has remained uncultivated for some time, Rhizobium microorganisms will be probably scarce in the soil. In such cases, it is a frequent practice that the seeds be inoculated before sowing, to assure the desired nodulation. Leguminous plants are considerably benefited as atmospheric nitrogen fixing nodules form, obtaining improved results and higher protein content. Besides, in nitrogen-deficient soils, benefits are obtained since their original contents do not exhaust.
There are many species and strains different from Rhizobium bacteria. Thus, specific strains can be identified for each crop, which will not necessarily make an effective nodulation in a different crop. Although most of the soils contain a natural rhizobial population, these wild strains are frequently ineffective for a desired nitrogen fixation. Therefore, the best way to assure an appropriate symbiosis with a certain crop is to inoculate either the seed or the soil, at the moment of the sowing, with bacteria of a selected rhizobian strain.
The well-known Rhizobium inoculant compositions contain the microbial strain together with an appropriate carrier medium.
Initially, inoculant compositions were used in agarized cultures and they were not very popular at that time as their practical use was very limited. Then, formulations with peat as carrier medium were used, which are still being used at present.
Coating seeds with an inoculant film is the most preferred method of inoculation. The inoculant is placed in intimate contact with the seeds in order to assure a quick formation of nodules in plants after germination. This can be achieved by means of the application of a powder composition of bacteria in dormant state, in a mixture with a powder carrier such as kaolin, peat, etc. Although a stable product is obtained, the powder does not completely stick onto the seeds, thus resulting in the loss of powder and therefore in the decrease of bacterial concentration in the coating, besides the problems in the sowing machines, such as blockage of pipes and other conducts which transport the seeds towards the soil.
Further on, oil-base inoculants appeared, which brought important practical advantages for their application, as compared to powder formulations, besides allowing the preparation of mixed formulations of inoculant and fungicides. However, and disadvantageously, in order to have a good survival of the bacteria in this type of formulations, it is necessary to store them at about 0xc2x0 C., which increases costs, since it requires to have cooling means, and maintain the cooling chain until the moment they are eventually used.
Recently, aqueous base inoculants were developed, which have the same practical use as the oil-based inoculants, an important advantage over them is that aqueous base inoculant can be stored at room temperature for long periods of time, without decreasing the number of viable bacteria.
It is known that the process of nitrogen fixation is activated by a nitrogenase enzyme which contains molybdenum (Mo). If molybdenum could be spread over the surface of the seed at the moment of germination, inoculating bacteria would achieve a quick nodulation and an immediate air nitrogen fixation, as well.
An important additional factor to keep in mind is the release of toxic compounds, usually called exudates, by the seeds. These exudates possess an inhibitory effect on bacteria, mushrooms, yeasts, etc. These effects may be also found in the leguminous seeds. The most important agents for toxic response were identified as condensed tannins in white clover (trifolium repens L.) for Rhizobium (Young and Paterson, 1980); for four strains of Rhizobium trifolii (T. Subterraneum L., T. Visiculosum Savi and T. Pratense L.) it is probably involved a reaction between the tannic acid of the seed and the iron of the medium (The Zamik and Wright, 1987); D""Arcy-Lameta A. (Study of soybean and lentil root exudates; Identification of some polyphenolic compounds. Relation with planlet physiology. Plant and Soil, 92, 113-123, 1986) have identified some polyphenolic compounds in diffusates of lentil seeds (Lens culinaris) and of soybean seeds (Glycine max L.). Others identified exudates were Myricetina (3, 5, 3xe2x80x2, 4xe2x80x2, 5xe2x80x2haexahidroflavone), desoxy-5flavones, isoflavonoids (Ali, F. S. and You Loymnachan, Inhibition of Bradyrhizobium japonicum by Diffusates from soybean seeds. Soil Biol. Biochem, 22(7), 973-976, 1990).
At the moment of inoculation, the inoculated seeds of leguminosae begin interacting with the bacteria over their surface (Rhizobium and Bradyrhizobium), thus producing a progressive death of said microorganisms (Bowden, G. D., The toxicity of legume seeds diffusate towards Rhizobia and other bacteria. Plant and Soil, 15 (2), 155-165, 1961).
In order to avoid these inconveniences, these exudates, such as the tannins, must be, somehow, neutralized by another compound having basic functions, which is inert for the beneficial bacteria and the seed/plant.
It has been found that the novel combination of a molybdenum compound and a compound that neutralizes toxic exudates of the seed in an aqueous base provides an inoculating composition which produces an immediate formation of nodules in the crown of the root of the germinated plants, and consequently nitrogen fixation, with which a strong plant with a high-protein performance is obtained from the beginning. The novel combination of a neutralizer compound of these toxic exudates with a compound of Mo on the surface of the seed, provides the root of the germinated plant with a high concentration of viable rhizobial bacteria and an availability of Mo independently of the characteristics of the soil. This novel combination provides performance results of plants inoculated with this composition, which are advantageously differed from those inoculated with the traditional formulations.
This invention provides an inoculating composition in an aqueous base for seeds which comprises at least a specie of microorganism which has a beneficial effect on plants, a Molybdenum compound and a polymer selected from those which have an amide, amine or imine moiety and appropriate exicipients.
Plants are leguminosae, with the preferable selected microorganisms being those of the Rhizobium or Bradyrhizobium genus.
A preferred polymer to be employed in this invention is polyvinyl pirrolidone (PVP), preferably in a concentration of about 0.2 to 5% weight/weight of the composition.
Preferred Molybdenum compounds are inorganic salts, preferably alkali metal salts, such as sodium molybdate and potassium molybdate. A more preferred salt is potassium molybdate, in a concentration ranging between 0.01 and 2.00% of the composition. More preferably, molybdenum compound is present in a concentration of 0.20%.
Furthermore, the invention provides a seed having a coating that comprises at least one species of microorganism having a beneficial effect on plants, a Mo compound and a polymer selected among those presenting an amide, amine or imine moiety.
The invention provides, also, a method for producing the immediate formation of nodules in the root of leguminous plants which comprises, coating the leguminous seeds with the above composition.
It is a further object of the invention to provide an inoculant liquid composition in an aqueous base, such as the one hereinabove described, that may be stored at room temperature for long time, while keeping the concentration of required viable bacterial for about two years.
The invention is carried out using bacteria of the Rhizobium and Bradyrhizobium genus, which are capable of forming nodules in the roots of one or more species of leguminous plants. Preferred strains include Bradyrhizobium japonicum, Rhizobium meliloti, Rhizobium leguminosarum biovar trifolii, Rhizobium leguminosarum biovar viceae and Rhizobium leguminosarum biovar phaseoli. 
The selection of Rhizobium and Bradyrhizobium strains effective for nitrogen fixation is a first necessary step for producing a high quality leguminous seed inoculant. Specificity of effectiveness is expressed as differences in action between species and differences among the species. Ecological factors should be also taken into account, such as competitive capacity for growing in the soil and in the rhizosphere, competitive ability for forming nodules and surviving in both, presence and absence of the plant. Other factors to be taken into account for selecting a microbial culture are tolerance to carrier means, tolerance to the substrate where it is applied and soil features.
After the desired rhiozobian strain is selected, it is cultured in a growth medium within a conventional fermenter. A suitable growth medium can be, for instance, an aqueous medium having saccharose, yeast autolysate, potassium phosphate, magnesium phosphate heptahydrate, calcium chloride and ferrous chloride. Fermentation is conducted preferably under shaking at about 30xc2x0 C., till a high bacteria concentration is achieved.
For practicing the inventive inoculating composition, shares of the fresh bacterial culture having viable cells are mixed with equal shares of a sterilized suspension containing a Molybdenum compound and a polymer of the type presenting an amide, amine or imine moiety, such as polyvinyl pirrolidone. Viable cells concentration used is that necessary for obtaining a concentration in the final composition of the invention of about 5.0xc3x97109 bacteria/ml.
Polymers having an amidic nitrogen may be those of the type having sub-units carrying a 2-pirrolidone cycle, such as polyvinyl pirrolidone (PVP), polymers of alkylated vinyl pirrolidone, vinyl pirrolidone/vinyl acetate, styrene and substituted derivatives copolymers.
Preferably, PVP K-60 is used, such as Luviskol K60 by BASF. Also alkylated vinyl pirrolidone polymers may be used (Agrimer AL, furnished by International Speciality Products).
A suitable range for concentration of PVP and the like in the inoculating composition of present invention may be about 0.2 and 5% weight/weight. Preferably. a 3% weight/weight concentration is used.
The suspension also contains suitable adjuvants that provide for stability into the final composition. As such adjuvants, stabilizers may be used such as carboxymethyl cellulose, Arabic gum, sodium alginate and the like. Preferably, sodium alginate is used in concentration ranging from 0.1 to 1%, more preferably about 5%.
Preferably, also carboxymethyl cellulose is used in a concentration of about 0.5 and 2%, more preferably at about 1%.
Furthermore, the composition may contain nutrients or growth factors, such as sugars, aminoacids, proteins, salts, and the like. Additionally, the composition may contain osmoregulating agents, buffers, etc.
The inoculating composition of the invention may be stored at room temperature for about two years, the bacteria survival being guaranteed. The inventive aqueous composition may be stored in 250 ml plastic bags, which are sterilized before filling and sealed after filling.
Coating of seeds may be performed by spraying or simply mixing the aqueous composition with the seeds in a hopper or suitable container till a uniform seed coating is achieved. The inoculant/seed proportion can be about 150 cm3 of the inoculating composition per 50 kilograms of seeds.
The invention has been generally described, and will be better understood when taken in reference to certain non limiting examples, which are included only for illustrative purposes.